B cells play a unique and important role in adaptive immunity by providing antibody-mediated protection against pathogens. It is critical that B cell activation be highly regulated in order to ensure appropriate immune response, and to prevent development of autoimmune diseases and B cell malignancies. At the heart of the B cell immune response is the B cell antigen receptor (BCR), which binds antigen and transduces activation signals by recruiting key signaling proteins into cell surface microclusters. It is becoming increasingly appreciated that proteins that operate at membrane-cytoskeleton interface exert exquisite control on BCR spatiotemporal organization and thus determine the strength of BCR signaling and humoral immunity. However, the contribution of myosin motor proteins towards regulation of B cell immunity remains poorly understood. In this application, we propose to investigate the role of Myo18a, a newly identified unconventional myosin motor protein, in B cell activation and immune response. We recently reported that BCR crosslinking leads to recruitment of the ? isoform of Myo18a to BCR signaling clusters. Our preliminary data show that B cell-specific conditional deletion of Myo18a in mice (Myo18a-/-) results in exaggerated antigen- specific antibody production. Both, Myo18a-/- B cells and B6 B cells in which Myo18a? is knocked down using siRNAs, undergo stronger activation upon BCR crosslinking. These data suggest that Myo18a? limits the BCR response to antigen. Myo18a? contains several protein-protein interaction domains, suggesting that it may act as a scaffold for appropriate localization and movement of BCR signaling complexes. Recruitment of Myo18a? to BCR clusters is accompanied by its association with ezrin, a member of the ERM family of plasma membrane-actin cytoskeleton crosslinkers. Myo18a? also spatially co-clusters with the conventional myosin Myo2a during B cell spreading on antigen-bearing surface. Given that ezrin and Myo2a are both known to control spatial organization and dynamics of BCR signaling clusters, our data suggest that Myo18a? collaborates with ezrin and Myo2a to regulate B cell activation. We hypothesize that Myo18a? restricts B cell activation and humoral immunity by orchestrating the spatial organization and dynamics of the BCR. We will test this hypothesis in two specific aims: (1) To determine the role of Myo18a in regulation of B cell activation and immune response. (2) To identify the molecular mechanisms employed by Myo18a? to regulate BCR signaling. We expect that our studies will reveal if Myo18a alters B cell antibody response in vivo, identify the specific molecular and spatial mechanisms employed by Myo18a to restrict B cell activation, and ascertain if Myo18a?- mediated restriction of B cell activation relies on is collaboration with ezrin and/or Myo2a. Collectively our findings will identify a previously unrecognized role for this unconventional myosin family protein in B cell immunity. Mechanistic insights from these studies will lay the foundation for in-depth structure-function analysis of Myo18a-dependent cytoskeletal regulation in B cell activation, and reveal new avenues for research and therapeutic development in diseases associated with aberrant BCR signaling.